epilepsy and tsc2 haploinsufficiency lead to autistic-like social deficit behaviors in rats

ORIGINAL RESEARCH

Epilepsy and Tsc2 Haploinsufficiency Lead to Autistic-Like SocialDeficit Behaviors in Rats

Robert Waltereit • Birte Japs • Miriam Schneider •

Petrus J. de Vries • Dusan Bartsch

Received: 21 July 2010 / Accepted: 16 September 2010 / Published online: 7 October 2010

� Springer Science+Business Media, LLC 2010

Abstract There is a strong association between autism

spectrum disorders (ASD), epilepsy and intellectual dis-

ability in humans, but the nature of these correlations is

unclear. The monogenic disorder Tuberous Sclerosis

Complex (TSC) has high rates of ASD, epilepsy and cog-

nitive deficits. Here we used the Tsc2?/- (Eker) rat model of

TSC and an experimental epilepsy paradigm to study the

causal effect of seizures on learning and memory and social

behavior phenotypes. Status epilepticus was induced by

kainic acid injection at P7 and P14 in wild-type and Tsc2?/-

rats. At the age of 3–6 months, adult rats were assessed in

the open field, light/dark box, fear conditioning, Morris

water maze, novel object recognition and social interaction

tasks. Learning and memory was unimpaired in naıve

Tsc2?/- rats, and experimental epilepsy did not impair any

aspects of learning and memory in either wild-type or

Tsc2?/- rats. In contrast, rearing in the open field, novel

object exploration and social exploration was reduced in

naıve Tsc2?/- rats. Seizures induced anxiety and social

evade, and reduced social exploration and social contact

behavior in wild-type and Tsc2?/- rats. Our study shows

that Tsc2 haploinsufficiency and developmental status epi-

lepticus in wild-type and Tsc2?/- rats independently lead to

autistic-like social deficit behaviors. The results suggest that

the gene mutation may be sufficient to lead to some social

deficits, and that seizures have a direct and additive effect to

increase the likelihood and range of autistic-like behaviors.

Keywords Tuberous sclerosis � Autism � Mental

retardation � Epilepsy � Animal models

Introduction

Autism spectrum disorders (ASD) are characterized by

qualitative abnormalities in reciprocal social interaction,

communication, and repetitive and stereotyped patterns of

behavior. Epilepsy is seen in about 30% of individuals with

ASD. Conversely, in epilepsy populations there is an ASD

prevalence of about 32%. In spite of this strong correlation,

there is so far little experimental evidence for any direct

causal relationship between seizures and ASD (Clarke et al.

2005; Spence and Schneider 2009).

Tuberous Sclerosis Complex (TSC) is caused by het-

erozygous mutation in either the TSC1 or TSC2 gene.

About 25% of individuals with TSC meet criteria for

Edited by Pierre Roubertoux.

Robert Waltereit, Birte Japs contributed equally.

Petrus J. de Vries, Dusan Bartsch—Joint senior authorship.

R. Waltereit (&) � B. Japs � D. Bartsch

Department of Molecular Biology, Central Institute of Mental

Health and University of Heidelberg, Mannheim Medical

Faculty, J 5, 68159 Mannheim, Germany

e-mail: [email protected]

R. Waltereit

Department of Psychiatry and Psychotherapy, Central Institute

of Mental Health and University of Heidelberg, Mannheim

Medical Faculty, Mannheim, Germany

M. Schneider

Department of Psychopharmacology, Central Institute of Mental

Health and University of Heidelberg, Mannheim Medical

Faculty, Mannheim, Germany

P. J. de Vries

Cambridgeshire & Peterborough NHS Foundation Trust,

Cambridge, UK

P. J. de Vries

Developmental Psychiatry Section, University of Cambridge,

Cambridge, UK

123

Behav Genet (2011) 41:364–372

DOI 10.1007/s10519-010-9399-0

classic infantile autism, about 50% for ASD, and 70–90%

have a lifetime history of epilepsy (Smalley 1998; Bolton

et al. 2002). Approximately 30–40% of TSC patients have

global intellectual disability (IQ \ 70) (Joinson et al.

2003). When the total TSC population is studied, epilepsy

shows strong correlations with intellectual disability and

ASD (Smalley 1998; Gomez et al. 1999; de Vries et al.

2007).

Animal models reduce the complex scenarios of neu-

ropsychiatric disorders to more simply defined variables

and are as such useful to study mechanisms of pathology

(Fisch 2007). To date, three TSC animal models, Tsc1?/-,

Tsc2?/- knockout mice and spontaneous mutation Tsc2?/-

(Eker) rats (Eker and Mossige 1961), have been analyzed

for the neurobiological basis of behavioral phenotypes.

None of the models exhibit spontaneous seizure activity.

Although there is substantial variation between the differ-

ent animals, all models express changes in learning and

memory with the Tsc1?/- mice showing deficits in social

behavior as well (Waltereit et al. 2006; Goorden et al.

2007; Ehninger et al. 2008). Taking together the current

human and animal data, results suggest that seizures may

not be necessary to cause ASD (de Vries and Howe 2007;

Goorden et al. 2007), and that there might be direct genetic

effects (Smalley 1998; de Vries and Howe 2007). How-

ever, social deficits have only been reported in one TSC

animal model (Goorden et al. 2007), and it is not known

whether seizures may be sufficient to cause social deficit

behaviors in TSC or animal models (Spence and Schneider

2009).

To examine the effect of TSC genotype and epilepsy on

learning and memory and social behaviors, we studied

Tsc2?/- (Eker) and wild-type rats. To examine the impact

of seizures during development, we treated wild-type and

Tsc2?/- rats with kainic acid (KA) injections at postnatal

day 7 (P7) and P14. All animals responded with a typical

crescendo-like status epilepticus that lasted many hours

(Sayin et al. 2004). Animals of all four groups (naıve wild-

type; naıve Tsc2?/-; epilepsy wild-type; epilepsy Tsc2?/-)

were analyzed for behavioral changes at the age of

3–6 months (Fig. 1).

Experimental procedures

Animals

Rats were housed under a 12 h/12 h day–night cycle. Only

male rats were used for experiments. Tsc2?/- (Eker) and

wild-type genotypes were determined by PCR (Rennebeck

et al. 1998). In all experiments, littermates with similar

distribution to Tsc2?/- (Eker) and wild-type were used. All

testing took place during the day phase. All experimental

procedures were performed according to permission from

local state authorities (Regierungsprasidium Karlsruhe).

KA-induced status epilepticus

KA monohydrate (Sigma, Deisenhofen, Germany) was dis-

solved in phosphate-buffered saline. At P7, male offspring

received an intraperitoneal injection with 3 mg/kg KA, and

at P14, a second injection with 4 mg/kg KA. The animals

were returned to the home cage with their mother. About

15 min after injection, a crescendo-like status epilepticus

started in all injected animals, and lasted for several hours.

For this study, always a Tsc2?/- parent was crossed with a

wild-type parent. During the first status epilepticus, there

was 17.5% mortality, during the second one, there was no

mortality. Of the animals surviving the epilepsy paradigm,

39% revealed the Tsc2?/- (Eker) mutation after genotyping;

of all naıve rats, 44% were Tsc2?/-. Late-onset seizures were

observed neither during animal care nor behavioral analysis.

Open field

The arena had an area of 52 9 52 cm2, a height of 45 cm

and was made of grey polyvinylchloride (PVC). Light

intensity was 50 lux. Movements were recorded with a

digital video camera and analyzed on a personal computer

using Biobserve Viewer (Biobserve, Bonn, Germany)

tracking software. Rearings were detected by light barriers

at a height of 11 cm. At the beginning of the 30 min ses-

sion, animals were placed in the center of the arena. Data

were analyzed as 5 min bins.

Light/dark-box

The apparatus had an area of 75 9 25 cm2, a height of

40 cm and was made of grey PVC. The dark compartment

had an area of 25 9 25 cm2, was separated from the light

compartment by a grey PVC wall with a 10 cm wide,

15 cm high alleyway and was covered by a grey PVC plate.

The light compartment was illuminated with 100 lux. Rats

were initially placed in the dark, closed compartment and

allowed to habituate for 1 min to the apparatus. The

alleyway was then opened, movements in the light

Fig. 1 Experimental design. Tsc2?/- and wild-type rats were chal-

lenged with status epilepticus by KA injections at P7 and P14. Naıve

rats had not undergone the epilepsy paradigm. Behavior was analyzed

at the age of 3–6 months

Behav Genet (2011) 41:364–372 365

123

compartment were recorded with a digital video camera for

10 min and analyzed manually by a trained observer.

Novel object recognition

Behavior was recorded with a digital video camera and

analyzed manually by a trained and blinded observer

(Schneider et al. 2008). Objects were made of metal or glass

and existed in duplicate. Rats were habituated to the open

field for 10 min, 24 h before behavioral testing. The test

consisted of an initial 3-min sample phase (P1) and a 3-min

discrimination phase (P2) that were separated either by an

intertrial interval of 15 min or by an interval of 24 h. During

P1, the rat was placed in the centre of the open field and

exposed to an unknown object (A). After cessation of P1 the

rat was returned to the homecage and the object was removed.

The rat was placed back in the open field after 15 min or 24 h

for object discrimination during P2 and was then exposed to

the familiar object (A0, an identical copy of the object pre-

sented in P1) and a novel test object (B). Exploration of the

objects (sniffing, touching and gnawing) was recorded during

P1 and P2. Sitting beside or standing on top of the objects was

not scored as object investigation. Testing for the 15 min

interval was done 1 day before the 24 h interval test and

different objects were used for both tests.

Fear conditioning and extinction

The apparatus and tracking system was as described earlier

(Waltereit et al. 2008), with the exception of using a

chamber designed for rats as context A, area 25 9 30 cm2,

height 33 cm (H10-11R-TC, Coulbourn Instruments,

Allentown, GA, USA). Context B was a standard home

cage without litter. The conditioned stimulus (CS) was a

5000 Hz sinus wave of 30 s duration. The unconditioned

stimulus (UCS) was a scrambled footshock of 0.5 mA and

1 s duration, which was applied during the last second of

the CS. Day 1: Habituation. 10 min exploration in context

A. Day 2: Conditioning in context A. 3 min exploration,

30 s CS-UCS, 3 min exploration, 30 s CS-UCS, 3 min

exploration. Day 3: Recall of conditioning. 6 min explo-

ration in context A, 1 h interval, 3 min exploration in

context B, 3 min exploration in context B with CS. Day 4:

Extinction. 3 min exploration in context A, followed by 15

times 30 s CS and 3 min exploration in context A. Day 5:

Recall of extinction. Recall of conditioning. 6 min explo-

ration in context A, 1 h interval, 3 min exploration in

context B, 3 min exploration in context B with CS.

Morris water maze

The pool had a diameter of 145 cm, a height of 50 cm and

was made from black PVC. Rats were trained in the water

maze with extra-maze cues (water made opaque by addi-

tion of 3 L milk, water temperature 26 ± 1�C,

10 9 10 cm2 platform with white rubber surface, height of

the platform 13 cm with its top surface 2 cm below water

level, maximum trial duration 120, 15 s on platform at the

end of trials). Illumination was 30 lux. Animals were

trained for 5 days three times a day with 2 h intervals

(Waltereit et al. 2006). After a total of 15 training trials, the

probe trial was performed with the platform removed

(probe trial duration = 60 s). During training trials, ani-

mals were assessed for the latency to escape to the hidden

platform. During the probe trial, animals were assessed for

the time spent in the four quadrants and for the frequency

with which they crossed the position of the platform in the

target quadrant (and the respective position in the three

non-target quadrants). Swim paths were recorded using the

EthoVision video tracking system (Noldus Information

Technology, Wageningen, The Netherlands).

Social interaction

The test was performed in the open field (Schneider et al.

2008). Social partners were 6 week old male Long-Evans

rats (Charles River, Sulzfeld, Germany). All animals were

habituated for 5 min to the arena 24 h before testing. The

experimental animal was first placed into the test arena and

was allowed to habituate for 1 min before the social partner

was introduced. The following behavioral elements were

videotaped and quantified by a trained and blinded obser-

ver only for the experimental rats: (A) Social behavior:

contact behavior, social exploration and approach/follow-

ing were scored as social behaviors. (1) Contact behavior:

contact behavior includes (a) grooming (chewing and

licking the partner’s fur) and (b) crawling over/under the

partner; (2) social exploration: (a) anogenital investigation

(sniffing or licking the anogenital area of the social partner)

and (b) non-anogenital investigation (sniffing at any part of

the partner’s body, except the anogenital area); (3)

approach/following: approaching or following the social

partner in the test arena. (B) Evade: running, leaping or

swerving away from the social partner. Evade, which is

normally defined as a defensive behavior in the context of

social play, was scored in the social interaction test as an

active withdrawal from social contact.

Statistics

Analyzes (two-way analysis of variance (ANOVA) or two-

way repeated measures ANOVA, respectively, followed by

Bonferroni post-hoc tests) were performed with SigmaStat

software (Systat Software, San Jose, CA, USA). Differ-

ences were considered statistically significant if P \ 0.05.

Graphical artwork was created with Prism (GraphPad

366 Behav Genet (2011) 41:364–372

123

Software, San Diego, CA; USA) and CorelDraw software

(Corel Corporation, Ottawa, Canada). Graphs always show

mean, standard error of the mean (SEM) and significant

results from the two-way ANOVA or two-way repeated

measures ANOVA, respectively. Significant results from

Bonferroni post-hoc tests are indicated in the graphs as

well. One asterisk in a graph represents P \ 0.05, two

asterisks P \ 0.01, three asterisks P \ 0.001.

Results

We first evaluated general behavioral parameters. Loco-

motor activity was assessed in the open field. There were

no differences in distance travelled over time (Fig. 2a, two-

way repeated measures ANOVA for experimental groups

[F(3,230) = 0.9826, P [ 0.05], time [F(5,230) = 190.2,

P \ 0.001] and interaction [F(15,230) = 0.3975, P [ 0.05]),

but Tsc2?/- rats showed less rearings than wild-type animals

(Fig. 2b, two-way ANOVA for genotype [F(1,46) = 4.320,

P \ 0.05], epilepsy [F(1,46) = 1,293, P [ 0.05] and inter-

action [F(1,46) = 0.07021, P [ 0.05]). Anxiety was ana-

lyzed using the light/dark-box. The latency to enter the light

compartment was longer in rats which had undergone the

epilepsy paradigm (Fig. 2c, two-way ANOVA for genotype

[F(1,52) = 0.5466, P [ 0.05], epilepsy [F(1,52) = 4.941,

P \ 0.05] and interaction [F(1,52) = 0.4554, P [ 0.05]).

Next, we examined novel object recognition and explora-

tion. All animals spent a higher proportion of time exploring

the novel objects during the recall phase, indicating that they

recognized the previously presented object, and there were

no differences between experimental groups after an interval

of 15 min (Fig. 3a, two-way ANOVA for genotype

[F(1,52) = 0.04816, P [ 0.05], epilepsy [F(1,52) = 0.1031,

P [ 0.05] and interaction [F(1,52) = 0.02086, P [ 0.05])

and an interval of 24 h (Fig. 3b, two-way ANOVA for

genotype [F(1,54) = 0.5534, P [ 0.05], epilepsy [F(1,54) =

0.001447, P [ 0.05] and interaction [F(1,54) = 0.08213,

P [ 0.05]). However, Tsc2?/- rats spent less time than wild-

type animals exploring novel objects during the acquisition

phase (Fig. 3c, two-way ANOVA for genotype [F(1,54) =

5.732, P \ 0.05], epilepsy [F(1,54) = 0.01435, P [ 0.05]

and interaction [F(1,54) = 0.5991, P [ 0.05]).

We then started a series of experiments to assess

learning and memory. Fear conditioning is a form of

classical conditioning. Our protocol tested both contextual

and auditory cue conditioning, and also analyzed extinction

of these memories. After conditioning with two CS-UCS

presentations, all rats demonstrated learning of both con-

textual (Fig. 4a, two-way ANOVA for genotype [F(1,51) =

0.04820, P [ 0.05], epilepsy [F(1,51) = 0.08456, P [ 0.05]

and interaction [F(1,51) = 0.9020, P [ 0.05]) and auditory

cue associations (Fig. 4c, two-way ANOVA for experi-

mental group [F(3,100) = 0.1168, P [ 0.05], auditory cue

[F(1,100) = 84.68, P \ 0.001] and interaction [F(3,100) =

0.1482, P [ 0.05]). There were no differences between

experimental groups. After extinction of the associa-

tions by presenting the CS without UCS for 1 h, rats

showed some reduction in both contextual (Fig. 4b, two-

way ANOVA for genotype [F(1,51) = 2.308, P [ 0.05],

epilepsy [F(1,51) = 0.3967, P [ 0.05] and interaction

Fig. 2 Tsc2?/- reduces exploratory behavior and developmental

epilepsy increases anxiety. a Distance travelled in the open field.

Wild-type naıve n = 16, Tsc2?/- naıve n = 13, wild-type epilepsy

n = 12, Tsc2?/- epilepsy n = 9. b Rearings in the open field. Wild-

type naıve n = 16, Tsc2?/- naıve n = 13, wild-type epilepsy n = 12,

Tsc2?/- epilepsy n = 9. c Latency to light in the light/dark-box.


n = 15, Tsc2?/- epilepsy n = 12. Data are expressed as mean and

(b, c) SEM. * P \ 0.05

Behav Genet (2011) 41:364–372 367

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[F(1,51) = 0.0000205, P [ 0.05]) and auditory cue condit-

ionings (Fig. 4d, two-way ANOVA for experimental group

[F(3,100) = 0.6257, P [ 0.05], auditory cue [F(1,100) =

47.11, P \ 0.001] and interaction [F(3,100) = 0.02162,

P [ 0.05]), but there were no differences between groups.

The Morris water maze tests spatial memory, a form of

hippocampus-dependent learning. There were no differ-

ences in latency to escape to the platform during training

trials (Fig. 5a, two-way repeated measures ANOVA for

experimental groups [F(3,700) = 1.390, P [ 0.05], trials

[F(14,700) = 52.61, P \ 0.001] and interaction [F(42,700) =

1.103, P [ 0.05]). During a probe trial after the last

training trial, we analyzed swimming in the target quad-

rant (Fig. 5b, two-way ANOVA for experimental group

[F(3,100) = 0.1293, P [ 0.05], target [F(1,100) = 22.07, P \0.001] and interaction [F(3,100) = 0.6895, P [ 0.05]) and

swimming over the target platform position (Fig. 5c, two-

way ANOVA for experimental group [F(3,54) = 0.2630,

P [ 0.05], target [F(1,54) = 34.55, P \ 0.001] and inter-

action [F(3,54) = 0.3329, P [ 0.05]). All animals had

learned to search in the target region, and there were no

differences between groups. Swim speeds during the probe

trial were without differences between groups (mean ±

SEM): Wild-type naıve 20.58 cm/s ± 1.33, Tsc2?/- naıve

20.19 cm/s ± 1.44, wild-type epilepsy 20.92 cm/s ± 1.16,

Tsc2?/- epilepsy 20.25 cm/s ± 0.43. Wild-type naıve

n = 8, Tsc2?/- naıve n = 7, wild-type epilepsy n = 8,

Tsc2?/- epilepsy n = 8. Two-way ANOVA for genotype

[F(1,27) = 0.6446, P [ 0.05], epilepsy [F(1,27) = 0.8616,

P [ 0.05] and interaction [F(1,27) = 0.9019, P [ 0.05].

Finally, we performed a series of experiments to assess

various social behaviors in the open field with a young

adolescent wild-type rat as social partner. Firstly, social

exploration was examined using anogenital and non-ano-

genital exploration and approach & follow behaviors.

Summary scores of these three social exploration tasks

showed that naıve Tsc2?/- rats had reduced social behav-

iors (Fig. 6a) and that epilepsy induced reduction in social

exploration behaviors in the wild-type rats to rates similar

to the naıve Tsc2?/- rats (Fig. 6a, two-way ANOVA for



P [ 0.05], significant Bonferroni post-hoc tests: epilepsy

within wild-type P \ 0.01, genotype within naıve

P \ 0.05). These differences were predominantly attribut-

able to reduction in non-anogenital exploration (Fig. 6b,

two-way ANOVA for genotype [F(1,27) = 0.9666, P [0.05], epilepsy [F(1,27) = 0.9666, P [ 0.05] and interaction

[F(1,27) = 1.223, P [ 0.05]) rather than to significant

change in anogenital (Fig. 6c, two-way ANOVA for




within wild-type P \ 0.001) or approach and follow

behaviors (Fig. 6d, two-way ANOVA for genotype [F(1,27)

= 3.073, P [ 0.05], epilepsy [F(1,27) = 1.073, P [ 0.05]

and interaction [F(1,27) = 1.115, P [ 0.05]). Next we

examined social evade and contact behavior. Social evade

is the active avoidance of contact with the social partner.

Naıve rats in both groups showed similarly low rates of

evade. Rats which had undergone the epilepsy paradigm

Fig. 3 Novel object recognition. a Novel object preference (15 min

interval). Wild-type naıve n = 15, Tsc2?/- (Eker) naıve n = 12,

wild-type epilepsy n = 17, Tsc2?/- (Eker) epilepsy n = 12. b Novel

object preference (24 h interval). Wild-type naıve n = 16, Tsc2?/-

(Eker) naıve n = 13, wild-type epilepsy n = 17, Tsc2?/- (Eker)

epilepsy n = 12. c Object exploration time (mean values from

animals used both 15 min and 24 h interval experiments). Wild-type

naıve n = 16, Tsc2?/- naıve n = 13, wild-type epilepsy n = 17,

Tsc2?/- epilepsy n = 12. Data are expressed as mean and SEM.

* P \ 0.05

368 Behav Genet (2011) 41:364–372

123

showed increased social evade (Fig. 6e, two-way ANOVA

for genotype [F(1,27) = 2.467, P [ 0.05], epilepsy [F(1,27)

= 15.97, P \ 0.001] and interaction [F(1,27) = 1.471,


within Tsc2?/- P \ 0.01). Contact behavior is a further

type of normal social interaction in rodents. Epilepsy also

reduced contact behavior in wild-type and Tsc2?/- rats

(Fig. 6f, two-way ANOVA for genotype [F(1,27) =

0.008507, P [ 0.05], epilepsy [F(1,27) = 9.642, P \ 0.01]

and interaction [F(1,27) = 0.1144, P [ 0.05], significant

Bonferroni post-hoc tests: epilepsy within wild-type

P \ 0.05).

Discussion

Our study revealed that neither the Tsc2?/- (Eker) muta-

tion, KA-induced status epilepticus at P7 and P14 nor

combination of these paradigms induced learning and

memory deficits in rats. However, both the Tsc2?/-

mutation and the epilepsy paradigm independently caused

autistic-like social deficit behaviors with reduced novel

object, environmental and social exploration behaviors in

the naıve Tsc2?/- rat, and increased anxiety, social evade,

reduced social exploration and contact behavior in Tsc2?/-

and wild-type rats after experimental epilepsy.

The epilepsy paradigm induced status epilepticus on two

separate occasions for several hours during development.

Late-onset seizures were observed neither during animal

care nor behavioral analysis. We did not assess the animals

by electroencephalography (EEG) or electrocorticography

(ECoG) to detect subtle or non-convulsive epileptic dis-

charges during adult age. However, because the epilepsy

paradigm did not induce any learning and memory deficits,

it seems unlikely that long-lasting or late-onset epileptic

activity confounded the other results.

The findings for the naıve Tsc2?/- rats are in agreement

with our previous study, which found no learning and

memory deficits in conditioned taste aversion, radial maze

and the Morris water maze (Waltereit et al. 2006). Sur-

prisingly, the experimental epilepsy paradigm did not

induce learning and memory deficits. This is in contrast to

a similar study which made also use of a status epilepticus

paradigm (Sayin et al. 2004). The latter study used

Fig. 4 Fear conditioning and extinction are not altered. a Freezing

after contextual conditioning. Wild-type naıve n = 16, Tsc2?/- naıve

n = 13, wild-type epilepsy n = 15, Tsc2?/- epilepsy n = 11.

b Freezing after extinction of contextual conditioning. Wild-type

naıve n = 16, Tsc2?/- naıve n = 13, wild-type epilepsy n = 15,

Tsc2?/- epilepsy n = 11. c Freezing after auditory cue conditioning.


n = 15, Tsc2?/- epilepsy n = 11. d Freezing after extinction of

auditory cue conditioning. Wild-type naıve n = 16, Tsc2?/- naıve

n = 13, wild-type epilepsy n = 15, Tsc2?/- epilepsy n = 11. Data

are expressed as mean and SEM

Behav Genet (2011) 41:364–372 369

123

Sprague–Dawley rats (Dr. Stafstrom, personal communi-

cation), whereas our rats were bred on Long-Evans back-

ground. Thus, strain differences may explain the discrepant

findings for wild-type rats. Learning and memory deficits

in rats are not an equivalent of global intellectual disability

in TSC patients. Nevertheless, it is surprising that the

combination of Tsc2 haploinsufficiency and developmental

seizures did not result in any learning and memory deficits.

Global intellectual deficit in TSC (de Vries and Prather

2007) is strongly associated with prolonged seizures,

infantile spasms (Joinson et al. 2003; O’Callaghan et al.

2004) and onset of seizures in the first year of life (Jozwiak

et al. 1998; Gomez et al. 1999; Bolton et al. 2002; Curatolo

et al. 2008). An explanation for the discrepancy between

these TSC patients and our model could lie in the duration,

type or developmental timepoint of the experimental sei-

zures. We therefore acknowledge the possibility that the

epilepsy paradigm used here may not have been of suffi-

cient duration or intensity to induce global intellectual

impairment, that KA-induced status epilepticus may not

model the pathology of infantile spasms adequately, and

that the time point of epilepsy induction could also have

been too late in development given, that P7 and P14 in rats

is later in brain development than seizures during the first

6–12 months of human life. These would be important

experimental parameters to modify in future studies.

Our experiments focused on social interaction and

anxiety-related behaviors rather than on communication or

repetitive and stereotyped patterns of behavior, the other

two diagnostic domains of ASD (Tordjman et al. 2007;

Viding and Blakemore 2007). Tsc2?/- rats displayed

reduced exploratory behaviors. In the open field, Tsc2?/-

rats exhibited less rearings, which can be interpreted as

reduced exploration of the environment, given that loco-

motor behavior was unchanged. Object exploration time in

the novel object recognition task was reduced. Naıve

Tsc2?/- rats also showed less social exploration. In a

similar pattern, Tsc1?/- KO mice showed less interaction

with a social partner and reduced nest building behavior,

interpreted as analogous to autistic-like behavior in patients

with TSC (Goorden et al. 2007). In contrast, Tsc2?/- KO

mice were not impaired in exploration and social behavior

tests, which was explained by a modifier gene hypothesis

(Ehninger et al. 2008). In the light/dark-box, wild-type and

Tsc2?/- rats which had undergone the epilepsy paradigm

expressed increased anxiety. The study by Sayin and col-

leagues which made similar use of KA-induced status

epilepticus during development, also described increased

anxiety in adult rats in the elevated plus maze (Sayin et al.

2004). The social interaction test is a test traditionally used

to study anxiety-related behavior in animals due to its

sensitivity to both anxiolytic and anxiogenic effects (File

2000; File et al. 2001; Irvine et al. 2001) and is therefore

thought to present a model of social anxiety in humans

(File and Hyde 1978). In humans, social anxiety and ASD

are often seen in conjunction (Moldin and Rubenstein

2006). Moreover, impaired reciprocal social interaction is a

core deficit in ASD. These tests are therefore recognized as

ASD phenotype tasks (Crawley 2007; Moy et al. 2007).

The epilepsy paradigm reduced social exploration in adult

rats. In addition, social evade and contact behavior tasks

also showed significant impairments induced by the epi-

lepsy paradigm. This reduction might thus be related to the

Fig. 5 No learning and memory deficit in the Morris water maze.

a Latency to platform during training trials. Wild-type naıve n = 15,

Tsc2?/- naıve n = 13, wild-type epilepsy n = 14, Tsc2?/- epilepsy

n = 12. b Time in quadrants during probe trial. Wild-type naıve

n = 15, Tsc2?/- naıve n = 13, wild-type epilepsy n = 14, Tsc2?/-

epilepsy n = 12. c Platform crossings during probe trial. Wild-type

naıve n = 8, Tsc2?/- naıve n = 7, wild-type epilepsy n = 8, Tsc2?/-

epilepsy n = 8. Data are expressed as mean and SEM

370 Behav Genet (2011) 41:364–372

123

increased anxiety-related response towards the unfamiliar

social partner, but it could also indicate a general perfor-

mance deficit in appropriate social behavior. Taken toge-

ther, both Tsc2 haploinsufficiency and epilepsy

independently lead to autistic-like social deficits. It is of

note that the nature of social deficits was not identical in

the Tsc2?/- and developmental epilepsy groups. In the

human ASD literature, there have been suggestions of

subtle phenotypic differences between ASD with and

without epilepsy. Children with ASD and epilepsy, for

instance, showed significantly reduced social interaction

with peers of a similar age (Turk et al. 2009). We suggest

that in TSC the neurobiological abnormalities caused by

gene mutation may be sufficient to lead to some autistic-

like social deficit behaviors, and that seizures may have a

direct and additive effect by inducing further social deficits

to increase the likelihood and range of autistic-like

behaviors.

The apparent dissociation between learning and memory

and social deficits is of further interest. The results suggest

the possibility of a differential threshold of vulnerability—

that is, fewer seizures may be required to induce social

deficits and that more, prolonged or developmentally ear-

lier seizures may be required to lead to learning and

memory deficits. Returning to ASD, our results suggest that

epilepsy in general may induce social, but not necessarily

learning and memory deficits in individuals who have ASD

or who are at risk of ASD. This may help to explain the

increased rates of ASD in epilepsy populations and the

significantly increased rates of ASD in those with genetic

syndromes associated with a high risk of ASD.

Acknowledgments This work was supported by research grants

from Tuberose Sklerose Deutschland to R.W. and Deutsche For-

schungsgemeinschaft SFB 636 to D.B. The authors would like to

thank Lena Wendler for excellent technical support and Dr. Mathias

Zink for help with an initial experiment.

Fig. 6 Tsc2?/- and developmental epilepsy lead to deficits in social

behavior. a Total social exploration (total of b–d). Wild-type naıve


epilepsy n = 8. b Anogenital exploration. Wild-type naıve n = 8,

Tsc2?/- (Eker) naıve n = 7, wild-type epilepsy n = 8, Tsc2?/-

(Eker) epilepsy n = 8. c Non-anogenital exploration. Wild-type naıve

n = 8, Tsc2?/- (Eker) naıve n = 7, wild-type epilepsy n = 8,

Tsc2?/- (Eker) epilepsy n = 8. d Approach and follow. Wild-type

naıve n = 8, Tsc2?/- (Eker) naıve n = 7, wild-type epilepsy n = 8,

Tsc2?/- (Eker) epilepsy n = 8. e Social evade. Wild-type naıve


epilepsy n = 8. f Contact behavior. Wild-type naıve n = 8, Tsc2?/-

naıve n = 7, wild-type epilepsy n = 8, Tsc2?/- epilepsy n = 8. Data

are expressed as mean and SEM. * P \ 0.05, ** P \ 0.01,

*** P \ 0.001

Behav Genet (2011) 41:364–372 371

123

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epilepsy and tsc2 haploinsufficiency lead to autistic-like social deficit behaviors in rats

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